PROJECTSUMMARY CanceristhesecondleadingcauseofmortalityintheUnitedStates,resultingin596,000deathsin2016alone1. In particular, approximately one in eight women2 and one in seven men3 will be diagnosed with breast and prostatecancer,respectively,duringhisorherlifetime.Unfortunately,positivemarginsinradicalprostatectomy (RP)andlumpectomyarestronglycorrelatedwithlocalreoccurrencethatnecessitatessecondarytreatment4?10. For both diseases, formalin fixed, paraffin embedded (FFPE) histology is the gold standard for margin assessment,butitsapplicationislimitedtopost-operativeevaluation.Frozensectionanalysis(FSA)allowsintra- operativemarginassessment,butitssensitivityisgenerallypoorcomparedtoFFPEhistologyduetosampling limitations and artifacts associated with FSA processing11?20. The inability of surgeons to confirm the completenessoftumorresectionsintra-operativelycouldleadtolocalreoccurrenceandsecondarytreatments thatareassociatedwithincreasedriskandcosttothepatient.Twophotonmicroscopy(TPM)hasdemonstrated promise for rapid cancer margin assessment21?26 because it enables visualization of nuclear size and density, reorganizationofthesurroundingextracellularmatrixandotherimportanthallmarksofcanceranalogoustothose presentinFFPEhistology27.Unfortunately,themajorityofpreviousTPMsystemsemployedtable-topscanners thatrestrictedimagingtodissected,exvivospecimens. Weproposethedevelopmentofanultra-compact,multimodalhandheldTPMprobeforvideorateimaging andassessmentofcancermargins.ThehandheldTPMprobewillleverageoptimizedstainingprotocolsandreal time virtual H&E rendering to assess the surface margins of resected specimens prior to dissection and with minimalpreparation.HandheldTPMimagingofthesurfacemargindirectlywouldovercomesamplinglimitations inherent to FSA, which typically generates histological sectioning planes that sample only a thin slice of the surfacemargin.Towardsfutureinvivosurgicalguidanceapplications,handheldTPMwillalsoenablevideorate fluorescence lifetime microscopy (FLIM) with simultaneous detection of intrinsic second harmonic generation (SHG) for imaging with molecular specificity but without the need for staining. This technology would be transferable to in vivo assessment of the surgical cavity, which can only be accessed with a handheld probe. ThediagnosticutilityofhandheldTPMwillbeassessedonresectedbreastandprostatespecimensusingboth extrinsic and intrinsic contrast and compared to the clinical standard of FFPE histology. Handheld TPM with rapidstainingandvirtualH&Ecouldbeaviableadjuncttopost-operativehistologythatwouldenablerealtime intra-operativeassessmentofresectedspecimens.HandheldTPMimagingwithFLIM/SHGcouldyieldintrinsic biomarkersthatwouldbedirectlytransferabletoinvivoimage-guidedsurgicalapplications.